Smallpox, which is caused by variola virus, is a highly contagious disease with a high fatality rate. A successful, worldwide vaccination campaign during the 1950s-l970s, using live vaccinia virus, resulted in eradication of smallpox. However, there remains a remote, but distinct possibility that large stockpiles of the virus may have been produced and stored as a part of bioweapons program in some countries or by terrorist organizations that are presently hostile to the United States. Smallpox poses a grave danger as an agent of biological weapon because of its highly contagious nature. Since vaccination against the disease stopped during early 1970s in the U.S., a large number of the young generation is unvaccinated and vulnerable to possible bioterrorist attacks. Although existing smallpox vaccine is relatively safe, it is not without serious medical complications including eczema vaccinatum, vaccinia necrosum, and encephalitis. Given the high frequency of AIDS patients who are immunodeficient, use of the current vaccine could result in a serious public health disaster. Therefore, it is imperative to consider developing a second-generation smallpox vaccine that is safer, yet as effective as the existing vaccine. Presently, variola virus is not available to perform additional research and the immune correlate of protection against either variola or vaccinia virus is largely unknown. Given these circumstances, a study is proposed with a long-term goal of developing a safer smallpox vaccine, with the following specific aims: (1) to characterize humoral and cellular immune responses against vaccinia virus in macaques previously immunized with the virus; (2) to evaluate immunogenicity and degree of attenuation of three recombinant vaccinia viruses derived from two different vaccinia strains (Western Reserve and Wyeth) in mice; and (3) to compare immunogenicity and safety of the newly generated vaccinia virus(es) with those of currently licensed smallpox vaccine strain in macaques previously infected with chimeric SlV/HIV -1 (SHIV). A successful completion of the proposed projects would allow better understanding of immune correlates of protection against vaccinia virus and could facilitate development of a safer smallpox vaccine.